Graft-Versus-Host-Disease Recruits Both Monocyte and Pre-Conventional Dendritic Cell-Derived Tissue Antigen Presenting Cells Independent Of CCR2 and CCR6

Graft-versus-host disease (GVHD) limits the broader application of allogeneic hematopoietic stem cell transplantation. In prior studies we defined roles for both host and donor-derived antigen presenting cells (APCs) in the activation of alloreactive donor T cells and in promotion of GVHD. While initial T cell activation in GVHD occurs predominantly in secondary lymphoid organs, we have consistently observed MHCII⁺ donor-derived APCs, including dendritic cells (DCs), in histopathologic GVHD lesions, frequently adjacent to infiltrating T cells, suggesting they have a role in local GVHD reactions. Donor-derived tissue APCs (t-APCs), including tissue-DCs (t-DCs) could activate donor T cells through indirect or cross-presentation of host antigens, produce chemokines that recruit other effectors, and elaborate inflammatory mediators or suppressors of inflammation. We first characterized t-DC subsets in the skin and bowel of GVHD-affected mice. 129 (H-2^b) hosts were irradiated and reconstituted with B6 (H-2^b) BM with or without CD4⁺ and CD8⁺ T cells to induce GVHD and analyzed mononuclear cells from skin and bowel approximately 4 weeks post transplant. In skin, both main dermal DC populations (CD11b⁺ and CD103⁺) were significantly increased in GVHD mice as compared to BM alone controls, though the ratios of CD11b⁺: CD103⁺ DCs were similar. In the bowel lamina propria, the ratios of CD11b⁺CD103^- to CD11b⁺CD103⁺ were increased in GVHD mice in the colon but were similar to that in BM alone controls in the small bowel. We next studied the roles of CCR6 and CCR2 in the recruitment of donor-derived APCs to skin and bowel. We transplanted mice with CCR6^-/- BM in competition with wild type (wt) BM and found that the contribution of each to skin and bowel APCs matched their contributions to myeloid hematopoiesis in BM, spleen and blood, indicating that CCR6 is not required. To study the role of CCR2 we first compared mice transplanted with either wt or CCR2^-/- BM with wt T cells. Despite having a profound reduction in blood monocytes, all skin and bowel t-APC subsets were present in CCR2^-/- recipients, indicating that CCR2 is not required for t-APC recruitment in contrast to its role in many other models of inflammation. However, CD103⁺ DCs were more prevalent relative to CD11b⁺ DCs, consistent with a pre-cDC origin. Despite monocytopenia, recipients of CCR2^-/- BM developed clinical GVHD; histology data is being analyzed and will be presented. To better define the contributions of CCR2 to t-APC recruitment and to determine monocyte versus pre-cDC origin of t-DCs, we transplanted mice with CCR2^-/- BM in competition with wt BM and compared ratios of BM and blood precursors (pre-cDCs and monocytes) to t-DC ratios. For CD103⁺ DCs, wt/KO ratios matched the ratios of general myeloid hematopoiesis and pre-cDCs, indicating a pre-cDC origin. For CD11b⁺CD103^- DCs, the ratio of wt/KO matched that in blood monocytes. We further subsetted CD11b⁺ t-DCs based on the expression of Ly6C, MAR1, CD64 and CD24, used to differentiate pre-cDC from mono-derived DCs in other organs, and did not identify any population with wt/KO ratios that did not match that of the general CD11b⁺ DC population, suggesting that most if not all CD11b⁺ t-DCs are of monocyte origin. Experiments are underway examining the role of CX3CR1 in t-APC recruitment and these data will be presented.